Phospholipids are amphipathic molecules which are insoluble in aqueous solution. Lipid vesicles are microscopic capsules composed of at least one phospholipid bilayer surrounding an aqueous fluid center, having a diameter of from 200 .ANG. to several microns. Lipid vesicles having a single lipid bilayer are termed unilamellar vesicles and classified as small unilamellar vesicles (SUV) or large unilameller vesicles (LUV), having diameters of about 200 to 500 .ANG. and about 500 .ANG. to several microns, respectively.
Phospholipid molecules each have a polar head group attached to a hydrophobic long-chain hydrocarbon tail. In aqueous solution, and under the appropriate conditions, biological lipids self-assemble into closed vesicles surrounded by at least one lipid bilayer. The resulting vesicle bilayer is composed of two layers of lipid molecules, the polar head groups contacting the aqueous solution inside and outside the vesicle forming the inner and outer surfaces. The hydrophobic tails are oriented away from the aqueous solution and disposed internally in the vesicle membrane.
Unilamellar and multilamellar vesicles of various sizes may be prepared from a variety of phospholipids by ethanol injection, cholate dialysis, ultrasonication, or by simple mechanical agitation.
Ethanol injection proceeds by injecting an ethanolic solution of phospholipid through a small bore needle into a rapidly stirred aqueous solution, spontaneously forming unilamellar vesicles which entrain small amounts of the solution.
In cholate dialysis vesicles are prepared by adding an aqueous solution of the substance to be encapsulated and sodium cholate to solvent-free phospholipid, resulting in a suspension from which vesicles are spontaneously formed after removal of the detergent.
Vesicles may also be prepared by ultrasonication; i.e., sonicating an aqueous phospholipid suspension using a titanium-tipped sonifier probe.
It is generally accepted that lipid vesicles can interact with cells by various mechanisms, notably by adsorption onto cell surfaces, by endocytosis, by fusion and by liquid transfer. Although the definition of these various events is straightforward, experimental evidence that unambiguously demonstrates these phenomena is difficult to obtain (Pagano and Weinstein, 1978; Post, 1980; Pagano et al., 1981b).
In adsorption the intact vesicle becomes attached to the cell surface without becoming internalized. This may occur through general electrical or polar attraction, by specific attraction to surface receptors, or by being bonded to antibodies which attach to specific sites on the cell.
Phospholipid vesicles may also be taken up by cells through endocytosis, wherein the vesicles encounter the cell wall, are encapsulated by a portion of the cell wall, and are drawn within the cell as encapsulated vesicles, the contents of which are released into the cell through intracellular degradation mechanisms of the vesicle bilayer.
Phospholipid vesicles may also release their contents into a cell through fusion, the process wherein the lipid bilayer surrounding the vesicle merges with the plasma membrane comprising the cell wall, also formed from lipids, concomitantly releasing the vesicle contents into the cell.
Lipid molecules may also transfer between vesicles and cells without direct association of the cell with the vesicle or its contents.
Vesicle-cell interactions have in the past been studied using vesicles which were labeled using encapsulated aqueous space markers, radio labels or fluorescent phospholipids, or combinations thereof. Examples of encapsulated markers include sugars, antibiotics, proteins, carboxyfluorescein, technetium and indium. Definitive interpretation of the results using these markers has been complicated, however, because the vesicles are typically leaky. The rate of marker loss varies considerably with factors such as lipid type, cell type, and the medium employed. Similar problems are encountered with certain radio labeled and fluorescent phospholipid markers. They also present the problems of phospholipids from the vesicle lipid bilayer exchanging with other phospholipids in vesicles or cell membranes.
It is an object of this invention to synthesize a reliable, easily measured, radio labeled nonexchangeable phospholipid marker which can be incorporated into phospholipid vesicles for use with in vitro and in vivo assays to produce quantitatively accurate results.
Recent developments in liposome biotechnology have resulted in the commercial use of phospholipid vesicles in in vitro immunodiagnostics. In addition, liposomes are currently being tested in preclinical trials as vehicles for a variety of chemotherapeutic agents in man, as particular liposome preparations and liposomes coupled to specific antibodies home to different organs on in vivo administration. These techniques are expected to be particularly advantageous in cases where the therapeutic agent carried within the vesicle has highly toxic effects, because minute quantities of the agent can be delivered to the tissue or organ on which it is to work without having the patient endure general exposure to large quantities of toxic substances. For the advantages of these methods to be fully realized, an easily detectable and stable gamma (.gamma.) emitting liposome marker is required.
Previously known radiolabelling techniques have used [.sup.3 H] and [.sup.14 C], individually or in combination. The use of these compounds with in vitro quantitative analysis has presented the problem of requiring special and extravagant preparation. The radiation emitted from these compounds is detectable only after prolonged processing of tissues, precluding in vivo measurements in live subjects. In addition, these compounds can physically transfer from liposomes to cells, resulting in ambiguous analyses of liposome-cell interactions.